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Researchers Make Breakthrough In Gene Transfer

, an antibiotic. They transferred this altered RNA into hamster kidney cells, which then were exposed to the antibiotic. The cells now had two reasons to die. If they hadn't acquired the viral RNA, they would be killed by puromycin, which prevents mammalian cells from making proteins. If they had, they would die from the toxic effects of the viral RNA, though not from the antibiotic.

A small proportion of the cells survived, however. These cells seemed to have acquired the viral RNA because they survived their contact with puromycin. But that RNA must have mutated so that it no longer was toxic to cells.

The researchers cultured the survivors and showed that they did indeed contain pieces of Sindbis RNA. They also found that this RNA was very efficient at making other hamster cells resistant to puromycin.

The researchers mapped the adaptive mutations and cloned the mutant RNAs for use as noncytotoxic gene expression vectors. They also made vectors that could be launched from DNA copies of the RNA. "The basic idea is that if you transfect cells with Sindbis vectors that have these adaptive mutations, Sindbis will establish replication, confer puromycin resistance, and the cells will be perfectly happy and capable of normal growth," Rice says.

The Sindbis vectors contain two regulatory regions. One regulates the gene for puromycin resistance; the other controls a foreign gene of choice. To test the effectiveness of the vectors, the researchers tried out several foreign genes, including luciferase, the enzyme that makes fireflies glow. Some of the genes were expressed at high levels, even after the host cells had divided 30 to 40 times.

In unpublished work, the group also has used these vectors to express proteins of hepatitis C virus, an important human pathogen for which no vaccine exists.

"These vectors should be very useful scientific tools beca
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Contact: Linda Sage
sage@medicine.WUSTL.edu
314-286-0119
Washington University School of Medicine
27-Oct-1998


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